Mutation, Mutagens,
and DNA Repair

Outline

We have been using the term 'mutation' pretty loosely up to this
point in the course...now we need to define it more precisely:
mutation-- a change in the genetic material (ie. DNA). We are
going to
spend some time talking about how mutations can occur and what their
consequences may be to cells; we will also be looking at the ways in
which cells avoid mutations by repairing DNA damage.

Why this focus? Why are mutations important? There are several
reasons: 1) they may have deleterious or (rarely) advantageous
consequences to an organism (or its descendants); 2) they are important
to geneticists: the most common way we study something is to break
it--ie., we search for or make a variant (mutant) lacking the ability to
perform a process which we want to study. These genetic variants possess
mutant alleles of the genes we are interested in studying. 3) Mutations
are important as the major source of genetic variation which fuels
evolutionary change (as we will see later when we talk about population
genetics and evolution).

Let's further define mutation as a heritable change in the
genetic material. This point becomes important in multicellular
organisms where we must distinguish between changes in gametes (germline
mutations) and changes in body cells (somatic mutations). The former are
passed on to one's offspring; the latter are not but we will see they can
be very important in causing cancer.

In detection of germline mutations in humans and measurement of
human mutation rates we have the problem of diploidy. Most forward
mutations (normal gene to mutant form) are recessive and so won't be
detected unless a zygote gets two copies of the mutant allele.
[Reversion or reverse mutation (mutant back to normal) is generally much
less frequent because there are a lot more ways to "break" a gene than
there are to reverse an existing mutation.] So how can we detect and
measure rates of new mutations? We can look at dominant mutations on
occuring on the autosomes and at both recessive and dominant mutations on
the X chromosome, since males are hemizygous for X-linked genes.
Example: achondroplasia occurs sporadically (in families with no previous
history) as a result of new mutations in the gene for the fibroblast
growth factor receptor. One study detected seven infants born with
sporadic achondroplasia in one year among 242,257 total births recorded.
So the rate (actually a frequency but we won't be concerned about the
difference for the purposes of thinking about rates in this course) is
7/242,257 x 1/2 (2 alleles per zygote) = 1.4 x 10e-5.

This rate is roughly in the middle of the range reported for
various human genes: those with high mutation rates like NF1
(neurofibromatosis type 1) and DMD (Duchenne muscular dystrophy) (ca. 1 x
10e-4) and those with low rates of new mutation like the Huntington's
Disease gene (1 x 10e-6). This hundred-fold range shows that mutation
rates per gene can be intrinsically different.

Why might this be? Two possible explanations are: 1) target
size and 2) hot spots. Some genes are large, meaning that there are many
bases at which mutations could alter or disrupt their function. The
large target argument could well be responsible for the high rates of
mutation of the NF and DMD genes, as these are known to have very large
protein coding regions. Alternatively, some genes may be in regions of
chromosomes which are more susceptible to genetic damage/change or may
contain sequences which are more likely to be altered by spontaneous
mutations; the achondroplasia gene is known to contain a hot spot of the
latter type (a CpG sequence, discussed below).

From studies like these in vivo and others using human cells in
vitro, the overall human mutation rate is estimated to be about 1 x 10e-6
per gene per generation. (Therefore the HD gene rate is probably more
typical than the other genes mentioned above.) This rate is similar
to those measured in various prokaryotic and eukaryotic microorganisms.
We can use the
estimated human mutation rate to determine its impact on the likelihood
of changes occurring in each generation: a rate of 1 x 10e-6
mutations/gene x 5 x 10e4 genes/haploid genome = 5 x 10e-2 mutations per
gamete (=5/100 or 1/20). 1/20 x 2 gametes per zygote = 1/10 chance that
each zygote carries a new mutation somewhere in the genome. This seems
like
a very high number but we need to remember that most mutations are
recessive and thus will not be expressed in the heterozygous condition.

Mutations, or heritable alterations in the genetic material, may
be gross (at the level of the chromosome, which we have already
discussed) or point alterations (this technically means mutations not
visible as cytological abnormalities and/or those which map to a single
"point" in experimental crosses). The latter can involve just a single
nucleotide pair in DNA. In this section, we will be considering small
changes in DNA, of the point mutation type.

A. Base pair (nucleotide pair) substitutions

These are of two types: transitions (purine to purine or
pyrimidine to pyrimidine) and transversions (purine to pyrimidine or
pyrimidine to purine). We break these down into the two categories
because they can occur in different ways.

The consequences of base substitution mutations in protein coding
regions of a gene depend on the substitution and its location. They may be
silent, not resulting in a
new amino acid in the protein sequence, eg. GCA or GCG codons in mRNA
both mean arginine [this is often true in the third position of a codon,
especially with transitions because of "wobble" base pairing]. A base
substitution could also result in an amino acid substitution; this is
referred to as a missense mutation. For example, CTC in the DNA
sense
strand [GAG in mRNA] will specify a glutamate residue in the protein;
this is altered to CAC in the DNA or GUG in the mRNA, resulting in a
valine residue in the beta-globin protein chain causing sickle-cell anemia.
Missense mutations may have very serious consquences, as in the case of
sickle-cell anemia, mild consequences as in the case of hemoglobin C (a
different amino acid substitution in position 6 of beta-globin) or no
phenotype as in the case of two known amino acid substitutions at
position 7 of beta-globin. Finally, base substitutions in a protein coding
region may mutate an amino acid codon to a termination codon or vice
versa. The former type, which results in a prematurely shortened protein
is referred to as a nonsense mutation. The effects of nonsense
mutations
are variable depending upon how much of the truncated protein is present
and is required for its function.

Base substitution mutations may also occur in promoters or 5'
regulatory regions of genes or in introns and may affect their
transcription, translation, or splicing. Many of the beta-thalassemias are
the result of these types of non-structural mutations that affect the
level of expression of the globin genes. All of the types of
mutation
described above have been observed in human globin genes. Their
consequences depend on what they do to the level of expression of the
gene product and/or on what amino acid substitution may have occurred and
where it is in the protein.

B. Frameshift mutations

These result from the insertion or deletion of one or more (not
in multiples of three) nucleotides in the coding region of a gene. This
causes an alteration of the reading frame: since codons are groups of
three nucleotides, there are three possible reading frames for each gene
although only one is used.

A mutation of this sort changes all the amino acids downstream and is
very likely to create a nonfunctional product since it may differ greatly
from the normal protein. Further, reading frames other than the correct one
often contain stop codons which will truncate the mutant protein
prematurely.

A. Definition and sources

A spontaneous mutation is one that occurs as a result of natural
processes in cells. We can distinguish these from induced mutations;
those that occur as a result of interaction of DNA with an outside agent
or mutagen. Since some of the same mechanisms are involved in producing
spontaneous and induced mutations, we will consider them together. Some
so-called "spontaneous mutations" probably are the result of
naturally occurring mutagens in the environment; nevertheless there are
others that definitely arise spontaneously, for example, DNA replication
errors.

B. DNA replication errors and polymerase accuracy

Mistakes in DNA replication where an incorrect nucleotide is added will
lead to a mutation in the next round of DNA replication of the strand
with the incorrect nucleotide.The frequency at which a DNA polymerase
makes mistakes (inserts an incorrect base) will influence the spontaneous
mutation frequency and it has been observed that different polymerases
vary in their accuracy. One major factor affecting polymerase accuracy
is the presence of a "proofreading" 3'-5' exonuclease which will remove
incorrectly paired bases inserted by the polymerase. This was shown in
vitro with purified DNA polymerases (those with 3'-5' exonucleases
make
fewer mistakes) and genetically by Drake with bacteriophage T4 mutants:
T4 has its own polymerase with a 3'-5' exo. Drake isolated
mutator mutants (which had a higher spontaneous mutation rate than
normal) and antimutator mutants (lower mutation rate than normal)
in the
polymerase gene and showed that the mutators had a higher ratio of
polymerizing to exonuclease activity than normal and that the
antimutators had a lower ratio.
These studies showed that the function of the 3'-5' exonuclease is to
prevent misincorporation during DNA replication and to prevent
mutations. Mutator mutants have since been isolated in other organisms
and have been shown to affect various components of the DNA replication
complex; alterations in a number of these proteins are likely to affect
the accuracy of the system.

C. Base alterations and base damage

The bases of DNA are subject to spontaneous structural
alterations called tautomerization: they are capable of existing in
two
forms between which they interconvert. For example, guanine can exist in
keto or enol forms.
The keto form is favored but the enol form can occur by shifting a proton
and some electrons; these forms are called tautomers or structural
isomers. The various tautomer forms of the bases have different pairing
properties. Thymine can also have an enol form; adenine and cytosine
exist in amino or imino forms. If during DNA replication, G is in the
enol form, the polymerase will add a T across from it instead of the
normal C because the base pairing rules are changed (not a polymerase error).
The result is a G:C to A:T transition; tautomerization causes transition
mutations only.

Another mutatgenic process occurring in cells is spontaneous base
degradation. The deamination of cytosine to uracil happens at a
significant rate in cells.

Deamination can be repaired by a specific repair process which
detects uracil, not normally present in DNA; otherwise the U will cause A
to be inserted opposite it and cause a C:G to T:A transition when the DNA
is replicated.

Deamination of methylcytosine to thymine can also occur.
Methylcytosine occurs in the human genome at the sequence 5'CpG3', which
is normally avoided in the coding regions of genes. If the meC is
deaminated to T, there is no repair system which can recognize and remove
it (because T is a normal base in DNA). This means that wherever CpG
occurs in genes it is a "hot spot" for mutation. Such a hot spot has
recently been found in the achondroplasia gene.

A third type of spontaneous DNA damage that occurs frequently is damage
to the bases by free radicals of oxygen. These arise in cells as a
result of oxidative metabolism and also are formed by physical agents
such as radiation. An important oxidation product is
8-hydroxyguanine, which mispairs with adenine, resulting in G:C to T:A
transversions.

Still another type of spontaneous DNA damage is alkylation, the
addition of alkyl (methyl, ethyl, occasionally propyl) groups to the bases
or backbone of DNA. Alkylation can occur through reaction of compounds
such as S-adenosyl methionine with DNA. Alkylated bases may be subject to
spontaneous breakdown or mispairing.

D. Spontaneous frameshift
mutations

Streisinger observed in the 1960's that frameshift mutations in
bacteriophages tended to occur in areas with "runs" of repeats of one
nucleotide.

Example:
5' AGTCAATCCATGAAAAAATCAG 3'
3' TCAGTTAGGTACTTTTTTAGTC 5'

He proposed that these frameshifts are the result of "slipped mispairing"
between the template DNA strand and the newly synthesized strand during
DNA replication. In the sequence above, a likely spot for frameshift
mutations to occur would be in the stretch of 6 A:T base pairs.
Subsequent studies with genes from other organisms, including humans,
have shown that runs of repeated nucleotides are indeed hotspots for
frameshift mutations.

A mutagen is a natural or human-made agent (physical or chemical)
which can alter the structure or sequence of DNA.

A. Chemical mutagens

The first report of mutagenic action of a chemical was in 1942 by
Charlotte Auerbach, who showed that nitrogen mustard (component of
poisonous mustard gas used in World Wars I and II) could cause mutations
in cells. Since that time, many other mutagenic chemicals have been
identified and there is a huge industry and government bureaucracy
dedicated to finding them in food additives, industrial wastes, etc.

It is possible to distinguish chemical mutagens by their
modes of action; some of these cause mutations by mechanisms similar to
those which arise spontaneously while others are more like radiation (to
be considered next) in their effects.

1. Base analogs

These chemicals structurally resemble purines and pyrimidines and
may be incorporated into DNA in place of the normal bases during DNA
replication:

bromouracil (BU)--artificially created compound extensively
used in
research. Resembles thymine (has Br atom instead of methyl group) and
will be incorporated into DNA and pair with A like thymine. It has a
higher likelihood for tautomerization to the enol form (BU*)

aminopurine --adenine analog which can pair with T or (less
well)
with C; causes A:T to G:C or G:C to A:T transitions.
Base analogs cause transitions, as do spontaneous tautomerization
events.

2. Chemicals which alter structure and pairing properties
of bases

There are many such mutagens; some well-known examples are:

nitrous acid--formed by digestion of nitrites (preservatives)
in
foods.
It causes C to U, meC to T, and A to hypoxanthine deaminations. [See
above for the consequences of the first two events; hypoxanthine in DNA
pairs with C and causes transitions. Deamination by nitrous acid, like
spontaneous deamination, causes transitions.

nitrosoguanidine, methyl methanesulfonate, ethyl
methanesulfonate--chemical mutagens that react with bases and add
methyl or ethyl groups. Depending on the affected atom, the alkylated
base may then degrade to yield a baseless site, which is mutagenic and
recombinogenic, or mispair to result in mutations upon DNA
replication.

3. Intercalating agents

All are flat, multiple ring molecules which interact with bases of DNA
and insert between them. This insertion causes a "stretching" of the DNA
duplex and the DNA polymerase is "fooled" into inserting an extra base
opposite an intercalated molecule. The result is that intercalating
agents cause frameshifts.

4. Agents altering DNA structure

We are using this as a "catch-all" category which includes a
variety of different kinds of agents. These may be:

--large molecules which bind to bases in DNA and cause them to be
noncoding--we refer to these as "bulky" lesions (eg. NAAAF)

--agents causing intra- and inter-strand crosslinks (eg.
psoralens--found
in some vegetables and used in treatments of some skin conditions)

--chemicals causing DNA strand breaks (eg. peroxides)

What these agents have in common is that they probably cause mutations
not directly but by induction of mutagenic repair processes (to be
described later).

B. Radiation

Radiation was the first mutagenic agent known; its effects on
genes were first reported in the 1920's. Radiation itself was discovered
in 1890's: Roentgen discovered X-rays in 1895, Becquerel discovered
radioactivity in 1896, and Marie and Pierre Curie discovered radioactive
elements in 1898. These three discoveries and others led to the birth of
atomic physics and our understanding of electromagnetic radiation.

1. EM spectrum

Visible light and other forms of radiation are all types of
electromagnetic radiation (consists of electric and magnetic waves). The
length of EM waves (wavelength) varies widely and is inversely
proportional to the energy they contain: this is the basis of the
so-called EM spectrum.

The longest waves (AM radio) have the least energy while successively
shorter waves and increasing energy are seen with FM radio, TV,
microwaves, infrared, visible, ultraviolet (UV), X and gamma radiation.
The portion which is
biologically significant is UV and higher energy radiation.

2. Ionizing radiation

X- and gamma-rays are energetic enough that they produce reactive ions
(charged atoms or molecules) when they react with biological molecules;
thus they are referred to as ionizing radiation. This term also includes
corpuscular radiation--streams of atomic and subatomic particles emitted
by radioactive elements: these are of two types, alpha- and
beta-particles [alpha are helium nuclei, 2 protons and 2 neutrons; beta are
electrons].

UV radiation is not ionizing but can react with DNA and other
biological molecules and is also important as a mutagen.

The units now used for ionizing radiation of all types are rems
(roentgen equivalent man): 1 rem of any ionizing radiation produces
similar biological effects. The unit used previously was the rad
(radiation absorbed dose). However, the effects of different types of
radiation differ for one rad unit: one rad of alpha particles has a much
greater damaging effect than one rad of gamma rays; alpha particles have a
greater RBE (relative biological effectiveness) than gamma rays. The
relationship between these units is that:

# rads x RBE = # rems

In addition to the energy type and total dose of radiation the
dose rate should be considered: the same number of rems given in a
brief, intense exposure (high dose rate) causes burns and skin damage
versus a long-term weak exposure (low dose rate) which would only
increase risk of mutation and cancer.

3. Sources of radiation

Natural sources of radiation produce so-called background
radiation. These include cosmic rays from the sun and outer space,
radioactive elements in soil and terrestrial products (wood, stone) and
in the atmosphere (radon). One's exposure due to background radiation
varies with geographic location.

In addition, humans have created artificial sources of radiation
which contribute to our radiation exposure. Among these are medical
testing (diagnostic X-rays and other procedures), nuclear testing and
power plants, and various other products (TV's, smoke detectors, airport
X-rays).

Taken together, our overall total average exposure from all
sources is about 350 mrem/year; the major contributor of which is from
radon exposure. See the graph on page 281 of your text for the breakdown.

4. Biological effects of radiation

Ionizing radiation produces a range of damage to cells and
organisms primarily due to the production of free radicals of water (the
hydroxyl or OH radical). Free radicals possess unpaired electrons and
are chemically very reactive and will interact with DNA, proteins, lipids
in cell membranes, etc. Thus X-rays can cause DNA and protein damage
which may result in organelle failure, block cell division, or cause cell
death. The rapidly dividing cell types (blood cell-forming areas of bone
marrow, gastrointestinal tract lining) are the most affected by ionizing
radiation and the severity of the effects depends upon the dose
received. The information below is based upon accidental exposures of
nuclear plant workers and victims of atomic bomb explosions such as those
in Hiroshima and Nagasaki:

sublethal dose (100-250 rems): nausea and vomiting early; 1-2 wk.
latent
period followed by malaise, anorexia, diarrhea, hair loss, recovery
(latency due to time it takes hematopoetic or other damage to show up)

lethal dose (350-450 rems): nausea and vomiting early; 1 wk. latent
period followed by above with more severe symptoms including internal
bleeding; a 50% chance of death [LD50 : dose at which half of exposed
individuals will die; ca. 400 rems for humans]. Death is due to blood
cell or gastrointestinal failure.

supralethal dose (>650 rems): nausea and vomiting early, followed by
shock, abdominal pain, diarrhea, fever and death within hours or days.
Death is due to heart or CNS damage.

For the affected tissues and organs, the number of destroyed cells and
the likelihood of their replacement determines the survival chances. The
long term effects include increased cancer risk and increased risk of
mutations
in one's offspring.

5. Genetic effects of radiation

Ionizing radiation produces a range of effects on DNA both
through free radical effects and direct action:

-breaks in one or both strands (can lead to
rearrangements, deletions, chromosome loss, death if unrepaired; this is
from stimulation of recombination)

-damage to/loss of bases (mutations)

-crosslinking of DNA to itself or proteins

The genetic effects of radiation were reported in 1927 in
Drosophila by Muller and in 1928 in plants (barley) by Stadler; both
showed that the frequency of induced mutations is a function of X-ray
dose. Their experiments revealed that there was a linear relationship
between X-ray dose and induced mutation level, that there was no
threshold or "safe" dose of radiation and that all doses are significant,
and finally, that "split dose" experiments showed that the genetic
effects of radiation are cumulative.

6. UV (ultraviolet)

UV radiation is less energetic, and therefore non-ionizing, but
its wavelengths are preferentially absorbed by bases of DNA and by
aromatic amino acids of proteins, so it, too, has important biological
and genetic effects.

UV is normally classified in terms of its wavelength:
UV-C (180-290 nm)--"germicidal"--most energetic and
lethal, it is not found in sunlight because it is absorbed by the ozone
layer;
UV-B (290-320 nm)--major lethal/mutagenic fraction of sunlight;
UV-A (320 nm--visible)--"near UV"--also has deleterious
effects (primarily because it creates oxygen radicals) but it produces
very few pyrimidine dimers. Tanning beds will have UV-A
and UV-B. To see a graphic representation of the wavelengths of UV and
ozone absorption, click here.

The major lethal lesions are pyrimidine dimers in DNA (produced
by UV-B and UV-C)--these are the result of a covalent attachment between
adjacent pyrimidines in one strand. This is shown here for a thymine-thymine dimer and here for a thymine-cytosine dimer.
These dimers, like bulky lesions from chemicals, block transcription and
DNA replication and are lethal if unrepaired. They can stimulate mutation
and chromosome rearrangement as well.

Because DNA damage occurs spontaneously and as a result to
ubiquitous environmental agents, most organisms possess some capacity to
repair their DNA and DNA is the only macromolecule which IS repaired by
cells. We can divide "repair" mechanisms into 3 categories:

damage removal--involves cutting out and replacing a damaged or
inappropriate base or section of nucleotides

damage tolerance--not truly repair but a way of coping with
damage so that life can go on

We will look at examples of each type of repair, the mechanisms,
the consequences of mutations in each, in both model organisms and in
humans.

A. Damage reversal

1. Photoreactivation

This is one of the simplest and perhaps oldest repair
systems: it consists of a single enzyme which can split pyrimidine dimers
(break the covalent bond) in presence of light. Click
here to see the photoreactivation reaction.

The photolyase enzyme catalyzes this reaction; it is found in many
bacteria, lower eukaryotes, insects, and plants. It seems to be absent
in mammals (including humans). The gene is present in mammals but may
code for a protein with an accessory function in another type of repair.

2. Ligation of single strand breaks

X-rays and some chemicals like peroxides can cause breaks in
backbone of DNA. Simple breaks in one strand are rapidly repaired by DNA
ligase. Microbial mutants lacking ligase tend to have high levels of
recombination
since DNA ends are recombinogenic (very reactive). A human known only by
the code name of 46BR was found to have mutations in both of her DNA
ligase I genes; she had poor growth, immunodeficiency, and
sun sensitivity and died at a young age of lymphoma. Fibroblast cells from
46BR are sensitive to killing by DNA damaging agents including ionizing
radiation. In addition, the rare hereditary disease Bloom syndrome
also somehow is involved with DNA ligase deficiency (although the
Bloom syndrome protein is a DNA helicase); patients' cultured cells have
high levels
of chromosome aberrations and spontaneous mutation.

B. Damage removal

1. Base excision repair

The damaged or inappropriate base is removed from its sugar
linkage and replaced. These are glycosylase enzymes which cut the
base-sugar bond.
example: uracil glycosylase--enzyme which removes uracil from DNA.
Uracil is not supposed to be in DNA--can occur if RNA primers not removed
in DNA replication or (more likely) if cytosine is deaminated (this is
potentially mutagenic). The enzyme recognizes uracil and cuts the
glyscosyl linkage to deoxyribose. The sugar is then cleaved and a new
base put in by DNA polymerase using the other strand as a template.
Mutants lacking uracil glycosylase have elevated spontaneous mutation
levels (C to U is not fixed, which leads to transitions) and are
hyper-sensitive to killing and mutation by nitrous acid (which causes C
to U deamination).

There are other specific glycosylases for particular types of DNA damage
caused by radiation and chemicals.

2. Mismatch repair

This process occurs after DNA replication as a last "spellcheck"
on its accuracy. In E. coli, it adds another 100-1000-fold accuracy to
replication. It is carried out by a group of proteins which can scan DNA
and look for incorrectly paired bases (or unpaired bases) which will have
aberrant dimensions in the double helix. The incorrect nucleotide is
removed as part of a short stretch and then the DNA polymerase gets a
second try to get the right sequence.

Human mismatch repair proteins have recently been identified and are very
similar to those of the prokaryote E. coli and the simple eukaryote
yeast
(this is an old invention of cells); mutations are found to be passed in
the germline of families with some types of inherited colon cancer
(HPNCC).

3. Nucleotide excision repair

This system works on DNA damage which is "bulky" and creates a
block to DNA replication and transcription (so--UV-induced dimers and
some kinds of chemical adducts). It probably recognizes not a specific
structure but a distortion in the double helix. The mechanism consists
of cleavage of the DNA strand containing the damage by endonucleases on
either side of damage followed by exonuclease removal of a short segment
containing the damaged region. DNA polymerase can fill in the gap that
results. Excision repair is shown here .

Mutants that are defective in NER have been isolated in many organisms
and are sensitive to killing and mutagenesis by UV and chemicals which
act like UV. Humans with the hereditary disease xeroderma
pigmentosum
are sunlight-sensitive, they have very high risks of skin cancers on
sun-exposed areas of the body and have defects in genes homologous to
those required for NER in simple eukaryotes. NER mutants in lower
organisms are UV-sensitive and have elevated levels of mutation and
recombination induced by UV (because they are unable to use the accurate
NER method to remove pyrimidine dimers and must use mutagenic or
recombinogenic systems).

C. DNA damage tolerance

Not all DNA damage is or can be removed immediately; some of it
may persist for a while. If a DNA replication fork encounters DNA damage
such as a pyrimidine dimer it will normally act as a block to further
replication.

However, in eukaryotes, DNA replication initiates at multiple sites and
it may be able to resume downstream of a dimer, leaving a "gap" of
single-stranded unreplicated DNA. The gap is potentially just as
dangerous if not more so than the dimer if
the cell divides. So there is a way to repair the gap by recombination
with either the other homolog or the sister chromatid--this yields two
intact daughter molecules, one of which still contains the dimer.

1. Recombinational (daughter-strand gap) repair

This is a repair mechanism which promotes recombination to fix the
daughter-strand gap--not the dimer--and is a way to cope with the
problems of a non-coding lesion persisting in DNA. The events of
recombinational repair are shown here . This
type of recombinational repair is generally accurate (although it can
cause homozygosis of deleterious recessive alleles) and requires a
homolog or sister chromatid. The
products of the human breast cancer susceptibility genes BRCA1 and
BRCA2 may be
involved in recombinational repair together with homologs of the yeast
RAD51 and RAD52 genes.

A second type of recombinational repair which is used primarily to repair
broken DNA ends such as are caused by ionizing radiation and chemical
mutagens with similar action is the non-homologous end-joining reaction.
This repair system is also employed by B and T cells of the immune system
for genetic rearrangements needed for their function. The Ku70, Ku80, and
DNA-dependent protein kinase proteins are needed for non-homologous
end-joining. Rodent cell lines with mutations in these genes are very
sensitive to killing by ionizing radiation and defective in immune system
rearrangement.

2. Mutagenic repair (trans-lesion synthesis)

An alternative scenario for a DNA polymerase blocked at a dimer is to
change its specificity so that it can insert any nucleotide opposite the
dimer and continue replication ("mutate or die" scenario). See the figure . We know
that this can happen in bacteria and think that it probably happens in
eukaryotes, though the mechanism is not well understood. This is a reason
why repair may sometimes cause mutations.

Ataxia telangiectasia is a human autosomal recessive hereditary
disease which causes several defects including about a hundred-fold
increase in cancer susceptibility. AT patients' cells in culture show
abnormalities including spontaneous and radiation-induced chromosome
breaks and sensitivity to killing by X-rays. (Ironically, the patients
also show extreme sensitivity to killing by X-ray doses intended to be
therapeutic for their cancers.) However, AT cultured cells do not show a
defect in repair of X-ray damage to their DNA; instead, unlike normal
cells, they continue to replicate their DNA even when it has been damaged
by X-rays. It is the failure to recognize DNA damage and respond
appropriately by halting the cell cycle until repair can occur that leads
to chromosome aberrations and death after X-ray in the AT patients.

The defect in AT is one in a cell cycle checkpoint, a decision
point that governs progression through the next phase of the cell cycle.
There are genetically controlled checkpoints that decide entry into a new
cell cycle (G0 to G1 point), the decision to replicate the DNA (G1 to S
point), and the decision to divide (G2 to M point). Mutations in the
checkpoint genes can lead to uncontrolled cell growth, ie. cancer.

Although AT itself is a rare condition, it has been estimated
that the frequency of heterozygotes with one AT mutation is about 1% in
the population. These individuals also have a higher cancer risk and
intermediate radiation sensitivity. Thus, screening by X-ray methods
(eg. mammography) may increase the chances of an AT heterozygote
developing cancer.